The biogenesis of tRNA from primary gene transcripts to their final functional form in E. coli and mammalian cells will be investigated and, in particular, the function of RNase P, a ribonucleoprotein essential for the processing of the 5' termini of tRNAs will be examined in detail. In vitro mutagenesis of cloned genes for E. coli tRNA1(Tyr) and the RNA moiety (M1 RNA) of RNase P will be used to provide appropriate mutants for the study of RNase P-tRNA precursor interactions. The hypothesis that the sequence UGAAU in M1 RNA forms hydrogen-bonded base pairs during enzyme-substrate complex formation with the sequence GTUCPu (common to all E. coli tRNA precursors) will be tested both in vitro and in vivo. Site specific mutagenesis will also be employed to elucidate: 1) the nature of other interactions of M1 RNA with the protein moiety (C5 protein) of RNase P and tRNA precursor substrates; and 2) the nature of the RNA processing site at the 3' terminus of the mature M1 RNA sequence and the functional properties of a promoter-like sequence at position -78 in the gene for M1RNA; and 3) the role of the dihydrouracil loop and stem in tRNA1(Tyr) in RNA processing events during the biosynthesis of this tRNA. The 3' flanking regions of the genes for both tRNA1(Tyr) (strong and weak suppressor derivatives) and M1 RNA will be exchanged using recombinant DNA techniques to examine the phenotypic effects on the upstream genes of the unique sequence organization of the flanking regions. The methodology originated by Agarwal and co-workers will be utilized to clone the gene for the protein moiety of E. coli RNase P to facilitate studies of the RNA-protein interactions in the RNase P complex and the phenotypes of various RNase P mutants. The gene coding for the RNA component of an RNase P-like activity from HeLa cell nuclei will be cloned and characterized to investigate structure-function relationships of this particular ribonucleoprotein and to compare the structure of this RNA with that of M1 RNA.